title: Amphetamine actions at the serotonin transporter rely on the availability of phosphatidylinositol-4,5-bisphosphate.
abstract: Nerve functions require phosphatidylinositol-4,5-bisphosphate (PIP2) that binds to ion channels, thereby controlling their gating. Channel properties are also attributed to serotonin transporters (SERTs); however, SERT regulation by PIP2 has not been reported. SERTs control neurotransmission by removing serotonin from the extracellular space. An increase in extracellular serotonin results from transporter-mediated efflux triggered by amphetamine-like psychostimulants. Herein, we altered the abundance of PIP2 by activating phospholipase-C (PLC), using a scavenging peptide, and inhibiting PIP2-synthesis. We tested the effects of the verified scarcity of PIP2 on amphetamine-triggered SERT functions in human cells. We observed an interaction between SERT and PIP2 in pull-down assays. On decreased PIP2 availability, amphetamine-evoked currents were markedly reduced compared with controls, as was amphetamine-induced efflux. Signaling downstream of PLC was excluded as a cause for these effects. A reduction of substrate efflux due to PLC activation was also found with recombinant noradrenaline transporters and in rat hippocampal slices. Transmitter uptake was not affected by PIP2 reduction. Moreover, SERT was revealed to have a positively charged binding site for PIP2. Mutation of the latter resulted in a loss of amphetamine-induced SERT-mediated efflux and currents, as well as a lack of PIP2-dependent effects. Substrate uptake and surface expression were comparable between mutant and WT SERTs. These findings demonstrate that PIP2 binding to monoamine transporters is a prerequisite for amphetamine actions without being a requirement for neurotransmitter uptake. These results open the way to target amphetamine-induced SERT-dependent actions independently of normal SERT function and thus to treat psychostimulant addiction.
authors.affiliation: Center of Physiology and Pharmacology, Medical University Vienna, A-1090 Vienna, Austria.
authors.forename: Florian
authors.initials: F
authors.lastname: Buchmayer
authors.forename: Klaus
authors.initials: K
authors.lastname: Schicker
authors.forename: Thomas
authors.initials: T
authors.lastname: Steinkellner
authors.forename: Petra
authors.initials: P
authors.lastname: Geier
authors.forename: Gerald
authors.initials: G
authors.lastname: Stübiger
authors.forename: Peter J
authors.initials: PJ
authors.lastname: Hamilton
authors.forename: Andreas
authors.initials: A
authors.lastname: Jurik
authors.forename: Thomas
authors.initials: T
authors.lastname: Stockner
authors.forename: Jae-Won
authors.initials: JW
authors.lastname: Yang
authors.forename: Therese
authors.initials: T
authors.lastname: Montgomery
authors.forename: Marion
authors.initials: M
authors.lastname: Holy
authors.forename: Tina
authors.initials: T
authors.lastname: Hofmaier
authors.forename: Oliver
authors.initials: O
authors.lastname: Kudlacek
authors.forename: Heinrich J G
authors.initials: HJ
authors.lastname: Matthies
authors.forename: Gerhard F
authors.initials: GF
authors.lastname: Ecker
authors.forename: Valery
authors.initials: V
authors.lastname: Bochkov
authors.forename: Aurelio
authors.initials: A
authors.lastname: Galli
authors.forename: Stefan
authors.initials: S
authors.lastname: Boehm
authors.forename: Harald H
authors.initials: HH
authors.lastname: Sitte
mesh_terms: D000661:Amphetamine / Q000494:pharmacology*; D057809:HEK293 Cells; D006801:Humans; D019269:Phosphatidylinositol 4,5-Diphosphate / Q000378:metabolism*; D015290:Second Messenger Systems; D050486:Serotonin Plasma Membrane Transport Proteins / Q000187:drug effects* / Q000235:genetics
chemical_list: D019269:Phosphatidylinositol 4,5-Diphosphate; D050486:Serotonin Plasma Membrane Transport Proteins; D000661:Amphetamine
keywords: amperometry; mass spectrometry; phosphoinositide; release; reuptake